Project Summary: The long-term objective of our project is to develop a comprehensive mechanistic understanding of the regulation of terminal erythroid differentiation in normal and diseased states. This is important because disordered or ineffective erythropoiesis is a feature of a large number of human hematological disorders, a major global health problem. Despite intensive efforts, the mechanistic understanding of regulation of terminal erythroid differentiation remains far from complete. The studies on regulation of erythropoiesis in the past have been primarily focused on growth factors, cytokines and transcription factors. As erythropoiesis is a complex process that requires tight regulation, it is important to explore the regulation of erythropoiesis by other mechanisms. The methylation status of DNA influences many biologic processes including cell differentiation. Recent studies identified important roles of TET protein-mediated 5- hydroxymethylcytosine production and DNA demethylation in cell differentiation. This application focuses on understanding the underlying molecular mechanisms for the global demethylation during terminal erythroid differentiation and the function of this event in erythroid biology with a tight focus on TET3. We anticipate that successful accomplishment of the proposed studies will lead to a better understanding of erythroid cell development and differentiation in general. Specifically we expect that our proposed studies should validate the newly identified novel epigenetic regulatory pathway in erythroid biology and provide the basis for future high impact research endeavors. As aberrant DNA methylation underlies many hematological diseases including the dyserythropoiesis of myelodysplastic syndromes, it is likely that our findings may also provide novel insights into these diseases.